Measurement of complement activation by biomaterials by means of complement convertase cleavage of peptide substrates

ABSTRACT

A process for determining complement activation due to contact between a biomaterial and a complement system, by incubating in vitro the biomaterial with the complement system and determining the formation of a complement convertase by using a substrate of the complement convertase and detecting substrate cleavage. The biomaterial after said incubation with the complement system may be separated therefrom and formation of a complement convertase may be determined with the separated biomaterial, the separated complement system, or both. The complement convertase may be Factor B convertase, C3 convertase or C5 convertase, and the substrate may be a labeled oligopeptide comprising an amino acid sequence corresponding to the cleavage site of the complement convertase. Suitable labels are dyes, fluorochromes, radioactive atoms or groups, and enzymes. Either classical or alternative pathway complement activation, or both, are determined. The complement system may be a non-clotting derivative of blood, blood plasma or blood serum, including fibrinogen depleted forms, anticoagulated forms, and derivatives containing thrombin inhibitor. Complement convertase substrates suitable for use in the process are also disclosed.

This is a U.S. National Phase Application of Application No.PCT/NL97/00030, filed Feb. 3, 1997, based on European Application No.962002333, filed Feb. 2, 1996.

TECHNICAL FIELD

The invention is in the field of diagnostics and relates to a newdiagnostic technique in medicine. More specifically it is concerned withthe measurement of activation of an immunologic system in blood, thecomplement system, when blood is contacting a foreign body surface(biomaterial).

BACKGROUND OF THE INVENTION

Medical devices are frequently used in contact with blood in bloodbanks, cardiovascular applications, organ replacement and vascularsurgery. These devices are made from plastics, metals or modified tissueand have in common that they activate the natural host defence mechanismof blood by a foreign body reaction (FIG. 1). One of the direct effectsof blood with such a foreign surface is clotting, which is prevented byanticoagulants. The other effect is activation of the immune system,which cannot be prevented pharmacologically.

This immune response is effected by the complement system, a number ofserum proteins consisting of components, activators, stabilizers andinhibitors. The complement system initiates chemotaxis and activation ofleucocytes, is essential for phagocytosis of microorganisms and iscapable of killing bacteria directly by inducing cell lysis. Animplanted foreign body surface could also be attacked by the complementsystem. In view of the wide-reaching biologic effects of the complementsystem, the consequences of uncontrolled complement activation would bedevastating (1-4). Continued activation of the sequence attractsleukocytes which release lysosomal enzymes as a byproduct ofphagocytosis, which in turn cause necrosis of normal tissue (5-8).

Normally, tight controls are in effect which regulate the complementsystem to protect host tissue. The cascade is naturally moderated by theinstability of the enzymes formed. Once a component is activated,failure to combine with its substrate within milliseconds cause it todecay. In addition, several plasma inhibitors are present to control thecascade. However, during the use of medical devices these regulatorymechanisms appear often inadequate due to the unnatural surface.Therefore, testing of complement activation by materials used for theconstruction of medical devices is needed to ensure the use of materialswith as low complement activation as possible.

THE COMPLEMENT SYSTEM (FIG. 2)

Activation of the complement system can occur via two distinctroutes--the classical and the alternative pathway. The end result ofcomplement activation is cytolysis, although this is probably not themajor function of complement. In the course of complement activation,biologically active factors are released. These factors enhance theimmune response by directing neutrophil migration, promoting immuneadherence, increasing vascular permeability, and interacting with otherinflammatory systems.

The classical pathway components are designated C1g, C1r, C1s, C4, C2,C3, C5, C6, C7, C8, C9. The alternative pathway components aredesignated Factor B, Factor D, Properdin, H and I.

Initiation of the classical pathway begins when antibody binds antigen.C1g binds the altered Fc region of IgG or IgM that has bound antigen.Upon binding, C1r activates C1s which initiates the activation unit bycleaving a peptide from both C4 and C2. C1s thus cleaves C4 into C4a andC4b and C2 into C2a and C2b. C2a binds to C4b forming C4b2a. C4b2a, theC3 convertase, is a proteolytic enzyme. It cleaves C3 into C3b, whichmay bind to the activating surface, and C3a which is released into thefluid phase (9). C3 convertase has the ability to cleave many C3molecules. This could result in the deposition of a large number of C3bmolecules on the activating surface. However, due to the labile natureof C3b, very few molecules actually bind. C4b2a3b, the C5 convertase, isformed when C3 is cleaved. C5 convertase, also an enzyme, can cleavemany C5 molecules into C5a and C5b.

The alternative pathway provides natural, non-immune defense againstmicrobial infections. In addition, this pathway amplifiesantibody-antigen reactions.

Alternative pathway recognition occurs in the presence of C3b and anactivating substance such as bacterial lipoprotein, surfaces of certainparasites, yeasts, viruses and other foreign body surfaces, such asbiomaterials (10-15). C3b originates from classical pathway activationand/or from natural spontaneous hydrolysis of C3. The resulting C3bbinds to the surface of the activating substance. In the presence ofmagnesium, Factor B binds to the C3b which is bound to the activatingsurface (16,17). Factor D then cleaves B, releasing the Ba fragment andforming C3bBb. Properdin stabilizes the C3bBb complex and protects itfrom decay. C3bBbP is the alternative pathway convertase. It also hasthe ability to cleave many C3 molecules. Cleavage of C3 results in theformation of C3bBb3b, the C5 convertase (18,19). This enzyme is alsostabilized by P to form C3bBb3bP. C5 convertase can cleave manymolecules of C5 into C5a and C5b.

The complement system has a positive feedback mechanism that amplifiesactivation. C3b produced from either pathway interacts with Factors B, Dand P from the alternative pathway (20). This interaction createsadditional C3 convertase to activate the membrane attack complex.

The membrane attack complex is common to both pathways. It begins withthe cleavage of C5 by C5 convertase generated during either classical oralternative pathway activation. When C5 is cleaved, C5a is released intothe fluid phase while C5b attaches to the activating surface at abinding site distinct from that of C3b. One molecule each of C6 and C7binds to C5b to form a stable trimolecular complex to which C8 binds.Then, up to 6 molecules of C9 can bind to C8 enhancing the effectivenessof the attack complex to induce membrane damage if the activatingsurface is a microorganism.

The significance of complement activation is not limited to membranedamage resulting from the attack complex. The active peptides releasedin the course of complement activation contribute to the immune responseby increasing vascular permeability and contraction of smooth muscle,promoting immune adherence, granulocyte and platelet aggregation,enhancing phagocytosis, and directing the migration of neutrophils (PMN)and macrophages to the site of inflammation (21-24).

The cleavage of C3 and C5 results in the release of two smallbiologically active peptides, C3a and C5a. The peptides act asanaphylatoxins. They amplify the immune response by causing the releaseof histamine, slow releasing substance of anaphylaxis (SRS-A), andheparin from basophils and mast cells. These substances increasecapillary permeability and contraction of smooth muscle resulting inedema and inflammation (25-27).

In addition to its role as an anaphylatoxin, C5a is a potent chemotacticfactor. This mediator causes the directed migration of PMN andmacrophages to the site of inflammation so these leukocytes willphagocytize and clear immune complexes, bacteria and viruses from thesystem.

In a process known as immune adherence, C3b or C4b deposited on asoluble immune complex or surface permit binding of complement receptorson PMN, macrophages, red blood cells and platelets (28,29). In thesecases C3b and C5b are considered opsonins as their presence results inmore effective phagocytosis.

LABORATORY MEASUREMENT OF COMPLEMENT PROTEINS

The following two techniques for assessing the complement system areknown.

1) Hemolytic techniques measure the functional capacity of the entiresequence--either the classical or alternative pathway.

2) Immunological techniques measure the protein concentration of aspecific complement component or split product.

HEMOLYTIC TECHNIQUES

In order for lysis to occur in a hemolytic technique, all of thecomplement components must be present and functional. Thereforehemolytic techniques can screen both functional integrity anddeficiencies of the complement system (30,31).

To measure the functional capacity of the classical pathway, sheep redblood cells coated with hemolysin (rabbit IgG to sheep red blood cells)are used as target cells (sensitized cells). These Ag-Ab complexesactivate the classical pathway and result in lysis of the target cellswhen the components are functional and present in adequateconcentration. To determine the functional capacity of the alternativepathway, rabbit red blood cells are used as the target cell.

The hemolytic complement measurement is applicable to detectdeficiencies and functional disorders of complement proteins, since itis based on the function of complement to induce cell lysis, whichrequires a complete range of functional complement proteins. Theso-called CH50 method, which determines classical pathway activation,and the AP50 method for the alternative pathway have been extended byusing specific isolated complement proteins instead of whole serum,while the highly diluted test sample contains the unknown concentrationof the limiting complement component. By this method a more detailedmeasurement of the complement system can be performed, indicating whichcomponent is deficient.

However, in order to induce deficiencies of complement proteins in serumfrom healthy individuals, which must be used to determinebiocompatibility, a very high extent of complement activation andconsumption is required. Therefore, in general the hemolytic techniquesare not sensitive enough to detect complement activation bybiomaterials. Some hemolytic techniques based on isolated componentswith a highly diluted test sample appeared to be more sensitive, buteven complement activation induced by 5 m² surface of a heart-lungmachine could marginally be detected with these methods (32).

IMMUNOLOGIC TECHNIQUES

Polyclonal antibodies were raised against different epitopes of the(human) C3, C4 an C5 complement factor. With these antibodiesradioimmunoassays were developed against the minor split products ofthese complement factors, which are particularly performed afterprecipitation of the native factor (33-36). Binding of the antibody withthe split product in competition with a known concentration of labeledsplit product could then be measured. Later on also (monoclonal)antibodies were raised to epitopes of the split products, rendering ahigher specificity. Nowadays, radio-immunoassays, ELISA's and radialdiffusion assays are available to detect complement split products.

In contrast to the hemolytic techniques, immunologic techniques providea high sensitivity to detect complement activation, since they allowmeasurement of split-product formation, while these split products areonly found at very low concentrations in blood from healthy individuals.Thus, clinically the measurement of the soluble split products C3a, C4aand C5 a in blood plasma has allowed a more distinct evaluation ofcomplement activation in patients (37). Later on the soluble form of theterminal complex (SC5b-9) was found a sensitive marker of complementactivation (38). For detection of in vivo complement activation thesetechniques are most suitable, particularly since blood samples can becollected in medium containing inhibitors of the complement system. Thusonly the complement activation formed in vivo is measured in thesubsequent assay.

However, these in vivo or clinical studies cannot be used to determinethe biocompatibility of biomaterials. Main problem during clinical useis that during application of biomaterials the complement system isactivated by a variety of material-independent factors, such as surgicaldamage of tissue, ischemia, blood-air contact, endotoxin and drugs whichalltogether dominate complement activation induced by the biomaterial.Thus, for pure biocompatibility testing in vitro studies are required,based on exposure of the biomaterial to isolated blood or bloodcomponents (usually plasma or serum). At this end difficulties arise.Starting with the isolation of blood from a donor, during preparation ofplasma or serum for the test, and during the test phase itself in thetest tube the complement system is activated and high concentrations ofsplit products are formed in plasma or serum. This high concentration ofsplit products dominates the split products eventually formed by thetest biomaterials during the test procedure. Thus, the sensitiveimmunologic techniques appear unsuitable for in vitro testing ofbiocompatibility. Moreover, it has been shown that to some biomaterialsthe split products adsorb to the surface. By the immunologic techniquesthese adsorbed split products are not detected. This leads to falsenegative appreciation of the test sample.

SUMMARY OF THE INVENTION

The invention provides a process for determining complement activationdue to contact between a biomaterial and a complement system, comprisingincubating in vitro the biomaterial with the complement system anddetermining the formation of a complement convertase by using asubstrate of the complement convertase and detecting cleavage of saidsubstrate. Preferably the biomaterial after said incubation with thecomplement system is separated therefrom and the determination of theformation of a complement convertase is carried out with the separatedbiomaterial, the separated complement system, or both.

The term "biomaterial" as used herein refers generally to any material,or product made thereof, which could come (or be brought) into contactwith biological fluids such as blood and may or may not activatecomplement.

Preferably, the complement convertase is selected from the groupconsisting of Factor B convertase, C3 convertase and C5 convertase. Morepreferably, the complement convertase is C5 convertase.

Although in principle any complement convertase substrate and anydetection method of cleavage of said substrate can be used, it ispreferred that the complement convertase substrate is a labeledoligopeptide comprising an amino acid sequence corresponding to thecleavage site of the complement convertase. More preferably the labeledoligopeptide is a labeled tripeptide having the general formulaLeu-Gly-Arg-Label, or Leu-Ala-Arg-Label, or Gln-Lys-Arg-Label, whereinLabel represents the label and the terminal amino group of theN-terminal amino acid may be blocked. The label in said labeledoligopeptide may be selected from the group consisting of dyes,fluorochromes, radioactive atoms or groups, and enzymes, and preferablyis a label which becomes (more easily) visible or detectable aftercleavage of the complement convertase substrate.

The process may be carried out in such a way that either classicalpathway complement activation, alternative pathway complementactivation, or both, are determined. Proper selection of reactionconditions, such as temperature, or the presence of a particular kind ofmetal ions (Ca⁺⁺ or Mg⁺⁺ ions), or directed suppression of a chosenpathway (e.g. using antibodies effective for that purpose) can be usedto restrict the test to a selected complement activation pathway.

The complement system used in the process may be any fluid containingconstituents of the complement system. In case the process is used todetermine biocompatibility of a biomaterial, the complement system usedmust contain an effective complement system, at least an activeclassical pathway complement system, an active alternative pathwaycomplement system, and preferably both. The complement system used maybe an artificial system, or a natural or semi-natural complement system,such as preferably a non-clotting derivative of blood, blood plasma orblood serum, including fibrinogen depleted forms, anticoagulated forms,and derivatives containing thrombin inhibitor.

Interference by other enzymes than the relevant complement convertasemay be reduced by carrying out the incubation of biomaterial andcomplement system and/or the substrate cleavage step in the presence ofinhibitors of said other enzymes.

The aim of the process may be to determine the complement activatingproperties of a biomaterial, in which case the complement system willusually be derived from blood known to have an active classical pathwaycomplement system, an active alternative pathway complement system, orboth, such as pooled normal blood.

Alternatively, the aim of the process may be to determine the complementresponse properties of a complement system, such as the blood of apatient suspected of having a complement deficiency, in which casebiomaterials with known complement activating properties are used.

The invention furthermore relates to complement convertase substratesfor use in a process as defined herein, in particular a labeledoligopeptide comprising an amino acid sequence corresponding to thecleavage site of a complement convertase, more particularly a labeledtripeptide having the general formula Leu-Gly-Arg-Label, orLeu-Ala-Arg-Label, or Gly-Lys-Arg-Label, wherein Label represents thelabel and the terminal amino group of the N-terminal amino acid may beblocked.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically the interaction between blood and a foreignbiomaterial. When blood is exposed to biomaterial, the contact system(Factor XII and cofactors) as well as the complement system (C3) will beactivated. The contact system leads to further activation of the kinin,fibrinolytic and clotting system. Activation of the complement systemleads to generation of chemotactic and membrane attack complementpeptides. The products generated trigger platelets and leucocytes torelease enzymes. If this process escapes the natural control, a wholebody inflammatory response may induce severe illness to patients.

FIG. 2 shows schematically the classical and alternative pathways of thecomplement system. The complement system is initiated through theclassical or alternative pathway. The classical pathway components C1,C2 and C4 are in particular formed after antibody-antigen recognition,the alternative pathway is initiated by C3b binding and stabilization ofC3Bb by factors P and D on any foreign body surface, such asbiomaterials. After C3 cleavage by this convertase, newly formed C3bwill then bind to the target surface, thus amplifying the reaction. Byboth pathways C3 and subsequently C5 is cleaved by the respectiveconvertases and the membrane attack complex is formed onto the targetsurface as well as soluble in blood. C3a and C5a fragments arechemotactic and activating for leucocytes.

FIG. 3a shows the substrate conversion results after incubation ofpolydimethylsiloxane (PDMS) in fibrinogen-depleted plasma (def PPP) fordifferent substrates. After incubation of PDMS in def PPP, the washedPDMS was incubated in BOC-Leu-Gly-Arg-pNA solution while def PPP wasmixed with H-Leu-Gly-Arg-pNA or Bz-Leu-Gly-Arg-pNA. Results arecorrected for background colour. Enzymatic activity was formed as shownby cleavage of the R-Leu-Gly-Arg-pNA substrates. The substitution of Rwith BOC was most effective on the PDMS surface, H- and Bz-substitutionwere effective in the plasma solution.

FIG. 3b shows the convertase activity formed onto the PDMS surfaceduring incubation at variable times in plasma. The PDMS was incubated ata variable time in def PPP, washed and then subjected toBOC-Leu-Gly-Arg-pNA.

FIG. 4a shows the effect of ethylenediamine tetra-acetic acid (EDTA)during substrate cleavage. According to the protocol PDMS was incubatedin defibrinated plasma and then removed. H- or Bz-substrate was thenadded to the plasma and incubated for 60 hours to develop colour. Duringthis second incubation new complement convertases could be formed,unless EDTA was added. EDTA did not prevent substrate cleavage by formedcomplexes. Cleavage of Bz-Leu-Gly-Arg-pNA was not reduced by EDTA, whichindicates aspecificity of this substrate.

FIG. 4b shows the effect of heating defibrinated plasma prior to theincubation. Def PPP was heated for 30 min at 56° C. before PDMS wasincubated therein. Factor B from the alternative complement pathway isknown to denaturate at temperatures above 56° C. By treatment of plasmaat that temperature the alternative pathway of complement becomesafunctional. If PDMS was incubated in heated plasma no furtheractivation of the complement system was observed.

FIGS. 5a,5b show the effect of different materials. PDMS(polydimethylsiloxane), PTFE (polytetrafluoroethylene) and PE(polyethylene) all showed binding of convertase on the surface (FIG. 5a,using BOC-Leu-Gly-Arg-pNA as substrate) and release in the incubationplasma (PPP) solution (FIG. 5b, using H-Leu-Gly-Arg-pNA as substrate).With the present test conditions the measurement in plasma appeared mostdiscriminative between the materials, showing PDMS as most activating.

FIG. 6 shows the effect of various inhibitors during the incubation.PDMS was incubated in defibrinated plasma aliquots which differed by theinhibitors added prior to incubation, i.e., EDTA, aprotinin, C1-esteraseinhibitor and thrombin inhibitor. EDTA almost completely preventedsubstrate conversion, aprotinin did not inhibit substrate conversion.This supports specificity of the substrate under these in vitroconditions for complement convertase activity, since aprotinin at theconcentrations in this test inhibits kallikrein, plasmin, and(chymo)trypsin.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides an enzymatic technique to determineconvertase activity towards synthetic peptides, which results in releaseof a labelling substance, such as a chromogenic, fluorescent, orradioactive label.

Although the complement system is activated through enzymatic cleavageof Factors B, C3 and C5 by convertases, these enzymes have not yet beenconsidered as useful markers for complement activation due to theirintrinsic lability, short half life, but most of all due to theiraspecific nature (39-42). Therefore it was a surprise to observeremaining activity of these convertases on the surface of materials, aswell as in the fluid phase, which could be ascribed to complementconvertase activity by using inhibitors of other potential substratecleaving enzymes.

The amino acid sequence of the natural substrates for the complementconvertases (Factors B, C3, C5) is known. Also the cleavage site ofFactor B by the Factor B cleaving enzyme C3bD, of C3 by theC3convertases (C4b2a and C3bBbp) and of C5 by the C5 convertase(C3bBb3bP) is known (43,44). Thus an artificial substrate resemblingthese structures could be used for these enzymatic processes. Thesesmall peptides substrates can be labeled in different ways (45,46) tomeasure the cleavage by the corresponding convertase activity. Asuitable labeled peptide substrate for C3-convertase would beLeu-Ala-Arg-pNA, for C5-convertase Leu-Gly-Arg-pNA, and for FactorB-convertase Gly-Lys-Arg-pNA. The nature of the label is not critical,and the amino group of the N-terminal amino acid may be free (H-form) orblocked (e.g. by BOC or Bz).

The new enzymatic technique is based on the convertases formed on thesurface of the test material and detects these convertases bound to thesurface and/or free in plasma. Specifically for the bound complementproteins this technique is most suitable, in contrast to the hemolyticand immunologic techniques. The new enzymatic technique shares theadvantage of the hemolytic techniques in being relevant to the realactivity of complement, since it measures important steps in thecascade. Also the new technique can reach the discriminating sensitivityof the immunologic techniques, since the existing convertase activity inplasma and on biomaterials is low, while it increases 2 to 5-fold afterincubation of the biomaterial in plasma. Since the convertase remainsactive during substrate conversion, the development of the colour can beextended until discrimination between the test samples is possible.Another important aspect of the complement measurement on biomaterialswith the presented in vitro technique is that substrate cleavage hasbeen shown for human as well as various animal species (38,39).

Peptide substrates have been described in the past for the measurementof proteases. The synthesis of labeled substrates has been described(43). Most of the activated plasma proteases are highly specific whencompared with digestive enzymes, such as trypsin or chymotrypsin. Insome cases, the plasma proteases cleave only one or two peptide bonds intheir natural substrates. This high enzyme specificity apparently is dueto the recognition of a specific amino acid sequence near the sensitivebond or a specific conformation of these amino acids near the sensitivebond.

A number of studies of the effect of the various blood coagulationfactors and complement enzymes have been published. Substratescontaining 4-nitroaniline and amides of 7-amino-4-methylcoumarin havebeen particularly useful. None of these substrates, however, arecompletely specific. Other problems are the low rate of enzymatichydrolysis and interference with other plasma proteins, such as albumin.

These disadvantages have made these substrate techniques for detectionof complement activation in vivo difficult to interprete, sincecomplement activation in vivo is accompanied by activation of otherblood cascade systems with some effect on substrate conversion.

The methodology described in this document enables a high specificity,mainly due to the fact that the biocompatibility will be determined invitro. In contrast to the measurements in blood samples from patients,during the in vitro measurement of biocompatibility, activation ofcascades other than complement can be suppressed with commerciallyavailable inhibitors, allowing the process to be controlled (38).Moreover, complement convertases have the specific property to bind tothe target biomaterial surface. Since the technique described herein isbased on incubation of the test material in plasma followed byseparation and washing of the test material, bound convertases on thematerial surface are separated from plasma prior to substrateconversion.

The present invention provides a technique to determine one importantaspect of the biocompatibility from biomaterials, being the extent ofcomplement activation. The present invention allows in vitrodetermination of complement activation by incubating biological fluids,blood or blood products with biomaterials, allowing the complementsystem to be activated and complement convertases to bind to thebiomaterials surface. Subsequently, the biomaterials are washed toremove unbound blood proteins and cells and then the biomaterials areincubated in medium containing specific substrate, allowing cleavage ofthis substrate by complement convertases. This cleavage can then bemeasured due to release of colour, fluorochromes, radioactive label,etc.

In general, substrates in the present invention are labeled tripeptides,although larger structures are not limited hereby. The substrates haveor comprise an amino acid sequence resembling the cleavage site of thenatural substrate for the complement convertase or any other amino acidsequence with low Km and high specificity. The concentration of thesubstrate in the test procedure will normally be in the order of μM inorder to have no substrate limitation during the test procedure.

Blood or blood plasma must be anticoagulated during incubation with thebiomaterials. Some anticoagulants, such as the Ca⁺⁺ depleting agentscitrate and EDTA, also affect the complement system. Mg⁺⁺ may be used inthese situations to allow alternative pathway complement activation.More preferably, serum or fibrinogen depleted plasma or blood/plasmawith specific thrombin inhibitors is used to prevent clotting duringincubation with biomaterials.

A technique described in the present invention may be used in thescreening of biomaterials to select the proper ones for construction ofmedical devices. It may also be used by test laboratories for bloodbiocompatibility testing. Further it may be used for research purposesduring clinical use of medical devices or inversily to test the abilityof an unknown blood sample to react with biomaterials.

The present invention also provides conditions to ensure specificity ofthe technique for complement convertases rather than other enzymes tocleave the substrates, by introduction of inhibitors for other enzymesduring substrate conversion.

The present invention comprises measurement of complement activationboth on the material surface and in the fluid phase. Dependent on thematerial surface, convertases are released in the biological fluid.

The present invention provides measurement of complement activation inhuman or animal biological fluid.

The present invention allows discrimination between activation of thealternative or classical pathway of the complement system, by employingCa⁺⁺ or Mg⁺⁺ in the biological fluid during incubation withbiomaterials, by means of heat treatment of the biological fluid or byinhibition of one of these pathways e.g. with antibodies.

The present invention enables measurement of complement convertasebinding to biomaterials during clinical use of these biomaterials byincubation of these used and washed biomaterials in the substratemedium.

The present invention allows measurement of convertase activity withsubstrates labeled with chromophores or fluorogenes or other suitablemarkers which are released during peptide cleavage of substrate by thecomplement convertase.

The present invention provides the possibility to characterizecomplement activation by any device, independent of three-dimensionalstructure or size. If needed the incubation time with substrate can beadjusted to the number of convertases formed during incubation whichmight be dependent on the surface area and material characteristics ofthe test material.

The present invention can also be used to detect any deficiencies of thecomplement system to respond to standardized material with well definedcharacteristics with respect to complement activation. Thesestandardized materials can be activators of the alternative or classicalcomplement pathway. The advantage of this technique over the existinghemolytic or immunologic techniques is that screening for complementdeficiency is possible without specialized laboratory techniques. Thepresent invention can even be developed into a bedside monitoring ofcomplement.

The present invention can be performed at varying temperatures, such asat room temperature or at 37° C., the main effect is the reaction speed.

The present invention allows a great extent of standardization for boththe measurement of biocompatibility from biomaterials with pooled normalserum and for the measurement of complement response from patientsplasma to standard biomaterials. For the biocompatibility measurementone batch of serum can be stored deep frozen in aliquots, for complementtesting one batch of uniform material can be used.

The invention will be illustrated by the following examples which merelyserve to illustrate the invention and not to limit it to the detailsshown. These examples demonstrate that we have evaluated a technique todetect the activation of the complement system, when a biomaterial is incontact with blood plasma. In our experiments it is shown that thebiomaterial binds complement convertase and releases these convertasesin plasma, which results in conversion of specific substrate. Soactivation of the complement system by biomaterials can now be measureddirectly in the fluid as well as on the surface.

In these experiments we have also shown that by means of this techniquea classification can be made between different biomaterials with regardto the complement-biocompatibility. Therefore this test may become apowerful tool to access this biocompatibility of biomaterials in theselection and testing of materials to be used in medical devices.

EXAMPLE 1

Human blood was collected from a healthy volunteer by vena puncture.Blood was mixed immediately with sodium citrate (0.316%, finalconcentration) to prevent clotting. Then blood was centrifuged (1100 xg)to obtain separation between plasma and blood cells. In our experimentswe used this citrated human plasma after treatment with reptilase tocoagulate fibrinogen. This plasma, deficient of fibrinogen (def PPP),contains the other coagulation components and can be mixed with Ca⁺⁺containing buffer to optimize complement activation. This def PPP wasstored in aliquots at -80° C. We diluted this in 50 mM TRIS buffer+33 mMCaCl₂ (pH 7.4) to a percentage of 20% prior to use. The biomaterial PDMS(polydimethyl-siloxane, silicon rubber) was cut into 1 cm² pieces. Forcleaning the biomaterial it was incubated at first in 70% ethanol, andthen rinsed in 0.9% NaCl. During the experiment the pieces ofbiomaterials were incubated in 500 μl diluted def PPP during 0, 10, 30,or 60 min at room temperature. After incubation, the biomaterials wererinsed 3 times with saline. Then the biomaterials were incubated inBOC-Leu-Gly-Arg-pNA, diluted in TRIS buffer +33 mM CaCl₂, during 60hours at room temperature in the dark. Simultaneously, also 100 μl defPPP, in which the biomaterials had been incubated, were mixed with 100μl of H-Leu-Gly-Arg-pNA or Bz-Leu-Gly-Arg-pNA, diluted in TRIS buffer,supplemented with 33 mM CaCl₂. After these 60 hours the OD was measuredat 405 nm in a spectrophotometer (microplate reader 3550 UV; Biorad,Richmond, Calif., USA).

These experiments showed colour formation releasing from PDMS withsubstrate BOC-Leu-Gly-Arg-pNA and in the incubated def PPP withsubstrates H-Leu-Gly-Arg-pNA and Bz-Leu-Gly-Arg-pNA (FIG. 3a).

Then, the effect of the incubation time of the biomaterial in plasma wasinvestigated.

It is shown that substrate conversion, expressed as OD 405 nm, increaseswhen the biomaterial is for a longer time in contact with plasma (FIG.3b), indicating more C5-convertase formation. Since in this experimentafter an incubation time of 30 min there was sufficient conversion, thisincubation time was chosen in further experiments as a standard time.

EXAMPLE 2

To verify the specificity of complement convertase activity, PDMS wasincubated with diluted def PPP supplemented with Ca⁺⁺ (allowingconvertases to be formed) or with EDTA (preventing convertaseformation). This experiment showed that EDTA completely preventedconvertase formation, indicating some specificity of substrate forcomplement activation.

In a second experiment the incubated def PPP was incubated in thepresence of Ca⁺⁺ as described above and 100 μl def PPP was collectedafter 30 minutes. Then this collected def PPP was incubated withH-Leu-Gly-Arg-pNA and Bz-Leu-Gly-Arg-pNA in the presence of Ca⁺⁺ or inthe absence of Ca⁺⁺ with EDTA. The formed convertases remained active inEDTA, while additional convertase activity was formed in the presence ofCa⁺⁺ during the incubation of activated def PPP with substrateH-Leu-Gly-Arg-pNA (FIG. 4a). This neoformation of convertases can beconsidered as an artifact. Thus the second incubation of def PPP withsubstrate must be performed in Ca⁺⁺ /Mg⁺⁺ chelating medium, like EDTA.Substrate Bz-Leu-Gly-Arg-pNA is considered non-specific for complementactivation, since EDTA did not inhibit further cleavage of thissubstrate.

A third test for specificity was the incubation of PDMS with def PPPheated during 30 min at 56° C., in order to eliminate factor B from thealternative complement pathway. Under these conditions convertaseformation was considerably reduced, indicating the importance offunctional factor B from the alternative complement pathway inconvertase formation by PDMS (FIG. 4b).

EXAMPLE 3

We have investigated the effect of different types of biomaterial, sincediscrimination between materials is one of the major goals of thistechnique.

We used the materials: PDMS, PE (polyethylene) and PTFE(polytetrafluoroethylene), which are all frequently used for medicaldevices. We have tested the effects of convertase formation in thesolutions and onto the biomaterials. The materials PDMS, PE and PTFEactivate the complement system, indicated by conversion ofBOC-Leu-Gly-Arg-pNA and H-Leu-Gly-Arg-pNA (FIG. 5a,b). PDMS appeared astronger activator than PE, whereas PTFE was a very weak activator ofcomplement.

EXAMPLE 4

A number of enzyme inhibitors was used to further specify the complementconvertase test of the incubated def PPP with substrateH-Leu-Gly-Arg-pNA. During incubation with PDMS in different aliquots aspecific thrombin inhibitor (I2581, Kabi), C1 esterase inhibitor(Sigma), the plasmin/kallikrein/chymotrypsin inhibitor aprotinin (Bayer)and EDTA was used. EDTA was the best inhibitor, followed by C1-esteraseinhibitor, then the thrombin inhibitor, then aprotinin (FIG. 6). Thisindicates that the main activity towards cleavage of the substageH-Leu-Gly-Arg-pNA is complement dependent.

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We claim:
 1. A process for determining complement activation due tocontact between a biomaterial and a complement system, comprisingincubating in vitro the biomaterial with the complement system anddetermining the formation of a complement convertase by using asubstrate of the complement convertase and detecting cleavage of saidsubstrate.
 2. A process as claimed in claim 1, wherein the biomaterialis separated from the complement system, after said incubating, toprovide a biomaterial fraction and a complement system fraction, bothfractions having complement convertase activity, and wherein thedetermining of the formation of complement convertase is carried outwith the biomaterial fraction, the complement system fraction, or both.3. A process as claimed in claim 1, wherein said complement convertaseis selected from the group consisting of Factor B convertase, C3convertase and C5 convertase.
 4. A process as claimed in claim 1,wherein said complement convertase is C5 convertase.
 5. A process asclaimed in claim 1, wherein said complement convertase substrate is alabeled oligopeptide comprising an amino acid sequence corresponding tothe cleavage site of the complement convertase.
 6. A process as claimedin claim 5, wherein said labeled oligopeptide is a labeled tripeptide isselected from the group of peptides consisting of Leu-Gly-Arg-Label,Leu-Ala-Arg-Label, and Gln-Lys-Arg-Label, wherein the label and theterminal amino group of the N-terminal amino acid may be blocked.
 7. Aprocess as claimed in claim 5, wherein the label in said labeledoligopeptide is selected from the group consisting of dyes,fluorochromes, radioactive atoms or groups, and enzymes.
 8. A process asclaimed in claim 1, wherein either classical pathway complementactivation, alternative pathway complement activation, or both, aredetermined.
 9. A process as claimed in claim 1, wherein said complementsystem is a non-clotting derivative of blood, blood plasma or bloodserum.
 10. A process as claimed in claim 1, wherein the interference byother enzymes than the complement convertase is reduced by carrying outthe incubation of biomaterial and complement system and/or the substratecleavage step in the presence of inhibitors of said other enzymes.
 11. Aprocess as claimed in claim 1 to determine the complement activatingproperties of a biomaterial, wherein said complement system is derivedfrom blood known to have an active classical pathway complement system,an active alternative pathway complement system, or both.
 12. A processas claimed in claim 1 to determine the complement response properties ofa complement system, wherein biomaterials with known complementactivating properties are used.